Asking Tough Questions

By Elizabeth Walker | 31 July

Triple-negative breast cancers account for just 15-20% of breast cancers, but a disproportionate number of deaths. A Nature Communications paper from the Kleer Lab, in close collaboration with other U-M Department of Pathology labs, has discovered a reason why and could lead to better outcomes for patients.

Celina Kleer, MD’s lab in the Rogel Cancer Center is occupied by staff and students with a range of experiences and talents, but a common goal – understanding difficult breast cancers. A physician-scientist, Kleer has spent the research side of her career at U-M focusing on aggressive breast cancer phenotypes such as inflammatory breast cancer and endoplasmic carcinomas. Her most recent challenge, triple negative breast cancers, are particularly difficult to treat. Unlike estrogen receptor (ER) positive breast cancers which can be treated with anti-hormonal treatment, or HER2 neu positive tumors which can be treated with anti-HER2 drugs, there’s no targeted therapy for triple negative breast cancers. They also tend to be more aggressive and metastasize more quickly. “The molecular basis of triple negative breast cancer is being unraveled but is not quite understood. It's important to understand the mechanisms that are underlying these tumors’ aggressive behavior so we can inhibit metastases for example,” Kleer explains.

An aspiring physician, Talha Anwar, had just completed his undergrad at Brown University and was working at the National Institutes of Health when he realized he wanted research to be a part of his career as well. “I wanted to go to a school that was strong both in clinical medicine but also really strong in basic science research,” he recalls. That desire led him to U-M’s MD/ PhD Medical Scientist Training Program (MSTP). This joint effort between U-M’s medical and graduate schools, allows students to graduate with both degrees after completing the curriculum over seven to eight full calendar years, including summer lab rotations.

Anwar first rotated through Kleer’s lab at the end of his first year of medical school in 2012 and recalls that Kleer was very influential in his decision to choose the Molecular and Cellular Pathology (MCP) program for the PhD portion of his training. “She really strong-armed me into going into pathology because the department has a really good way that it encourages collaboration and there are so many Principal Investigators,” he says. Kleer echoes this sentiment saying that, with so many strong cancer research labs in U-M’s department, pathology is an excellent option. However, many students mistakenly believe that if they want to pursue a career in cancer research, it means that they should choose the cancer biology program. With his first two years of medical school complete, Kleer as his mentor, and a tightly-knit group of fellow MCP students and their PI’s to collaborate with, Anwar settled into his work in the lab and a focus on a protein called Enhancer of Zeste Homolog 2, or EZH2.

EZH2 is a quite well-studied protein, and the wealth of knowledge about EZH2 has exploded in the past few years. Anwar, Kleer, and their colleagues, already knew that EZH2 is an oncogene, meaning it causes cancer and that when the protein is removed, it will stop that cancer from being so aggressive. It was also known that the prevalence of EZH2 correlates with survival. So, women with higher levels of the protein in their cancer tend to do worse overall and have lower survival rates.  What’s interesting about triple-negative breast cancers is that there’s a considerable amount of genomic instability allowing for many mutations to occur. The lab was interested in trying to understand what the function of EZH2 was in this specific context.     

In its normal development, EZH2 is thought of as a transcriptional repressor, meaning that it prevents the transcription, or copying, of a protein by binding to another protein at a specific site. However, this wasn’t the case in triple-negative breast cancers. EZH2 levels are really high in these cancers because it’s an oncogene. Take a closer look at the mark of its function, and more questions arise. Anwar explains, “It’s called eH3k27me3, or histone3 lyphsene27 tri-methyl mark. The basic idea is that it’s a mark of EZH2 function, an epigenetic mark. EZH2 is an epigenetic protein.” There’s an inverse correlation between EZH2 levels and EZH2 function in this set of cancers, but why?

“It’s really puzzling because it suggests that there could be some other mechanism that EZH2 is using so we started to look at different ways in which EZH2 is regulated and we focused in on a specific phosphorylation site, P38,” Anwar says. Work done in the Kleer lab by a previous graduate student, Heather Moore, had found that EZH2 is modified in multiple different ways. One of these ways is a post-translational modification called phosphorylation, a biochemical process in which a phosphate is added to an organic compound. Moore’s work found that EZH2 can be phosphorylated as a specific residue.  So, Anwar had his questions. What was happening at P38? How does it relate to the discrepancy between EZH2 levels and function? How does it relate to breast cancer progression and metastasis?

Before these questions could be answered, Anwar would have to develop some tools to study the phosphorylation site. He’d also need to turn to other labs to collaborate. “We had to do things like making an antibody for this specific site. We had to do some cloning to make different mutations to do some cell biology assays,” he explains. This work took a couple of years and then, working with the histology core, they used the antibody he developed against the specific phosphorylation site and started staining cells. “We stained normal cells, breast cancers, all different types of cancers,” Anwar says. “Celina is a pathologist by training and so what she noticed was that tumor cells seemed to have this interesting staining pattern where EZH2 is in the cytoplasm of these cells.” Normally, these cells are in the nucleus. Something was happening to cause EZH2 to move from the nucleus to the cytoplasm. Was this significant? What was EZH2 doing there? Could this explain the inverse relationship between EZH2 levels and its function?

These were risky questions to ask and they couldn’t be answered by the Kleer lab alone. Because the MCP program encourages so many interactions between its students and mentors, finding collaborators happened very organically. “You get to know people really well and know what they’re versed in. When you collaborate with people you already know, there’s a certain level of trust that allows things to move faster,” Anwar says. He felt fortunate to have experts on hand. He turned to fellow student Jim Ropa, a member of Andrew Muntean, PhD’s Lab, for advice on taking a proteomics approach and to help analyze data. Additional bioinformatics and proteomics support came from the lab of Alexey Nesvizhskii, PhD. Sierrah Grigsby and her mentor, and MCP program director, Zaneta Nikolovska-Coleska, PhD, were consulted for their expertise in protein-protein interactions.   

“This is the paper where I have with the most collaborations in pathology,” Kleer says. “We discovered a new mechanism that enables triple-negative breast cancers to move, to invade, and to metastasize without much change in the tumor growth. So, it's a mechanism that mainly triggers metastasis,” she explains. Phosphor EZH2 binds to vinculin. Vinculin is a really important protein in focal adhesion so it allows the cells to migrate and invade. So now, the lab is interested in knowing how the binding of EZH2 and vinculin lead to vinculin activation. If they inhibit vinculin activation, could they prevent or block migration invasion? With more research, this discovery could lead to the development of biomarkers, and to testing new potential treatments.

A proactive approach and important therapeutic advance might be to develop small molecules that could block the binding in P38 and EZH2, inhibiting the entire oncogenic pathway and blocking the cascade of metastasis. It’s possible that this could work not only in triple negative breast cancers but in other cancers as well. The Kleer lab is already working with Nikolovska-Coleska trying to develop a strategy and writing grants to address this.

Kleer feels that the study brought her closer to her colleagues and says that while it can sometimes be a struggle for physician-scientists to feel they fit in, it’s not a problem at U-M. “I do feel that I can fit very nicely with my clinical colleagues and also with the basic scientists. The MCP program is getting stronger as we are interacting at different levels that foster collaboration.”

Anwar defended his thesis on this work in May and is now finishing his last two years in medical school. He believes he made the right decision in choosing U-M for his training, recalling, “I had a great experience in the lab and a great experience in the program.”

 
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